Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.

Alternative splicing of mRNA precursors provides an important means of genetic control and is a crucial step in the expression of most genes. Alternative splicing markedly affects human development, and its misregulation underlies many human diseases. Although the mechanisms of alternative splicing have been studied extensively, until the past few years we had not begun to realize fully the diversity and complexity of alternative splicing regulation by an intricate protein-RNA network. Great progress has been made by studying individual transcripts and through genome-wide approaches, which together provide a better picture of the mechanistic regulation of alternative pre-mRNA splicing.

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Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches

http://sites.utoronto.ca/intron/blencowecell2006.pdf

Alternative Splicing: New Insights from Global Analyses

We used cross-linking and immunoprecipitation coupled with high-throughput sequencing to identify binding sites in 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein that, when mutated, causes amyotrophic lateral sclerosis. Massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs were changed (including Fus (Tls), progranulin and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events were detected (including in sortilin, the receptor for progranulin) following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels were most depleted by reduction in TDP-43 were derived from genes with very long introns and that encode proteins involved in synaptic activity. Lastly, we found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3' untranslated region of its own transcript, thereby triggering nonsense-mediated RNA degradation.

/pdf/long-pre-mrna-depletion-and-rna-missplicing-contribute-to-4ijz4ygdlo.pdf

Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43

The SR protein family comprises a number of phylogenetically conserved and structurally related proteins with a characteristic domain rich in arginine and serine residues, known as the RS domain. They play significant roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. In addition they participate in post-splicing activities, such as mRNA nuclear export, nonsense-mediated mRNA decay and mRNA translation. These wide-ranging roles of SR proteins highlight their importance as pivotal regulators of mRNA metabolism, and if these functions are disrupted, developmental defects or disease may result. Furthermore, animal models have shown a highly specific, non-redundant role for individual SR proteins in the regulation of developmental processes. Here, we will review the current literature to demonstrate how SR proteins are emerging as one of the master regulators of gene expression.

The SR protein family of splicing factors: master regulators of gene expression.

Mounting evidence suggests that transcription and RNA processing are intimately coupled in vivo, although each process can occur independently in vitro. It is generally thought that polymerase II (Pol II) C-terminal domain (CTD) kinases are recruited near the transcription start site to overcome initial Pol II pausing events, and that stably bound kinases facilitate productive elongation and co-transcriptional RNA processing. Whereas most studies have focused on how RNA processing machineries take advantage of the transcriptional apparatus to efficiently modify nascent RNA, here we report that a well-studied splicing factor, SC35, affects transcriptional elongation in a gene-specific manner. SC35 depletion induces Pol II accumulation within the gene body and attenuated elongation, which are correlated with defective P-TEFb (a complex composed of CycT1-CDK9) recruitment and dramatically reduced CTD Ser2 phosphorylation. Recombinant SC35 is sufficient to rescue this defect in nuclear run-on experiments. These findings suggest a reciprocal functional relationship between the transcription and splicing machineries during gene expression.

/pdf/the-splicing-factor-sc35-has-an-active-role-in-5akdk9mwwc.pdf

The splicing factor SC35 has an active role in transcriptional elongation

Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing

Neuronal differentiation involves extensive reprogramming of gene expression. Many neuronal-specific genes are actively repressed in nonneuronal cells, while many others are induced in response to cell differentiation cues. Together these constitute the transcriptome in neurons to instruct specific neuronal functions (Rosenfeld et al. 2006). The transcriptome in neurons is further diversified by alternative splicing, arising from the expression of a number of neuronal-specific RNA-binding splicing regulators (Black and Grabowski 2003). In this issue of Genes & Development, Boutz et al. (2007b) report a novel switch in the expression of a pair of related splicing regulators that occurs during neuronal differentiation. These proteins, known as polypyrimidine tractbinding proteins (PTB) and neural PTB (nPTB), are structurally and functionally similar, but PTB is widely expressed in nonneuronal cells, and nPTB is restricted to neurons. Remarkably, ∼25% of neuronally induced alternative splicing events detected by mRNA isoform-sensitive splicing arrays are estimated to result from the down-regulation of PTB, and the up-regulation of its neuronal-specific cousin nPTB.

/pdf/ptb-nptb-switch-a-post-transcriptional-mechanism-for-15jg4a1eqg.pdf

PTB/nPTB switch: a post-transcriptional mechanism for programming neuronal differentiation.

Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication.

/pdf/maase-an-alternative-splicing-database-designed-for-1itxqjw4gp.pdf

Gabriela Coutinho-Mansfield

Papers

The splicing factor SC35 has an active role in transcriptional elongation

Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing

PTB/nPTB switch: a post-transcriptional mechanism for programming neuronal differentiation.

MAASE: an alternative splicing database designed for supporting splicing microarray applications.